1. Field of the Invention
The present invention relates to a method and device for establishing a white blood cell count in biological fluids in the presence of interfering substances.
2. Description of the Background Art
A white blood cell (WBC) count is one of the most commonly tested parameters in clinical laboratories. A laboratory WBC usually requires 5-10 ml. of venous blood, and the patient may have to wait up to three days for the laboratory results. Generally, methods for estimating WBC in biological fluids are based on automated cell counting technologies, in which the sample is diluted, and cells of different sizes and shapes are counted in a flow cell (U.S. Pat. Nos. 2,656,508; 3,502,973; 6,159,740). Instruments based on flow technology are expensive and require professionally trained personnel for running in a clinical laboratory environment. New analyzers using nanotechnology for flow cells are much smaller than the traditional cell counting instruments, but they are still in the development stage. Also, the complicated mechanical pump and valves systems required limit the options for manufacturers to offer a low cost instrument.
There are many instances in which rapid measurements of white blood cell count is important. Rapid measurements of the white blood cell count may be useful in a physician""s office, where clinicians use these measurements to assess the effects of therapeutic drugs, cytostatic medications, and certain infections. Patients who are on chemotherapy also need to check their white blood cell count frequently to ensure that they are eligible for the next treatment, which may be a problem if they live at a distance from their physician or a hospital. Schizophrenic patients taking Clozapine must monitor their white blood cell counts weekly. Patients suffering from chronic infections may have white blood cell counts in excess of 10,000 cells per microliter, and should be monitored. Being able to monitor members of a large population after a radiological accident or terrorist attack would facilitate triage and planning for the best use of medical resources.
Mastitis is an inflammation of the mammary gland in an animal""s udder that costs the dairy industry close to $2 billion annually in lost revenues. When a dairy animal suffers from clinical mastitis, her udder is visibly swollen or the milk is water, thick, or ropy. Unfortunately, an apparently healthy animal can harbor sub-clinical mastitis, which accounts for up to about 70% of the mastitis in dairy herds. Researchers have made many attempts to devise a test for this xe2x80x9cinvisiblexe2x80x9d mastitis. A somatic cell count (SCC) of milk, which consists of over 90% white blood cells, has been universally adopted as the measure of a mastitis infection. To date, the California Mastitis Test (CMT) is the most common test for field use. However, the CMT is labor intensive, and the test suffers from a subjective interpretation by the individual user and an unacceptably high false negative rate. There is still no suitable field test that can estimate WBC count increases resulting from sub-clinical mastitis.
For purposes of the present invention, xe2x80x9cdairy animalxe2x80x9d means any animal from which milk can be obtained. Nonlimiting examples of dairy animals are cows, sheep, goats, camels, and buffalo (bison). Tests for white blood cells for cows can be used for other types of dairy animals as well. Thus, when the present specification uses cows as an example, the process is not limited to cows but is applicable to all types of dairy animals.
In an agricultural setting, an accurate cow-side milk test for white blood cell count means potentially large savings for farmers, who can discard the milk from mastitis infected cows before it is pooled with, and hence contaminates, the milk from uninfected cows. Estimation of WBC using a colorimetric method is very desirable to detect sub-clinical mastitis.
Enzymes located on the walls of white blood cells have esterolytic activity. Various colorless chromogenic esters known in the art may be cleaved by this enzymatic hydrolysis, resulting in the formation of a colorless acid component and a color forming alcohol or phenol component. The color intensity can be measured quantitatively by means of a calorimeter or semi-quantitatively using a visual color chart. Berger et al., in U.S. Pat. No. 4,278,763, took advantage of this esterase property and developed dipsticks capable of detecting as little as 200 cells per microliter in human urine. A number of patents have issued following this first report (U.S. Pat. Nos. 4,637,979; 4,657,855; 4,716,236; 4,806,423), and urine dipsticks for white blood cell counts have been commercialized for many years.
Unfortunately, the efficacy of assays for analytes in a biological fluid sample can be reduced by the presence of interfering substance. For example, the colorimetric WBC dipstick technologies developed for human urine are not applicable to other biological fluids, such as whole blood and milk. The intense red background color of the red blood cells in a sample of whole blood masks the color developed during the enzymatic reaction. Likewise, the complex matrix of a milk sample contains interfering substances that dramatically inhibit the enzymatic reaction. With respect to urine samples, it would be desirable to improve the dipstick performance, including reducing test time and increasing sensitivity, by circumventing the adverse effects of interfering substances.
U.S. Pat. Nos. 5,463,745 and 6,010,866, describe a method for determining an analyte in a biological fluid sample in the presence of a substance which interferes with an assay for the analyte. However, this method must be implemented using analyte specific antibodies, which makes the separation method expensive.
A number of workers have developed filters which remove or deplete white blood cells from a sample, including those described in U.S. Pat. Nos. 6,337,026; 6,221,264; 5,938,940; 5,795,483; 5,783,094; 4,880,548; 5,258,127; 5,728,306; and 4,936,998. None of these filters was disclosed for use in conjunction with white blood cell count estimations.
Diagnostic kits and methods for counting white blood cells have also been reported. U.S. Pat. No. 6,046,019 discloses a method and device that involves multiple steps performing cell lysing, filtering, substrate addition, incubation, and reading. Semi-quantitative results were reported, but this device is difficult to use.
U.S. Pat. No. 5,700,645 teaches a method for separating and concentrating cells from milk and other biological samples. This patent addresses the need for concentrating cells so as to increase the sensitivity and to separate bacterial cells for various assay. However, there is nothing in this patent about estimating white blood cell counts in the milk.
Accordingly, there is a need to develop a method and device which uses the recognized advantages of the colorimetric detection of white blood cells while providing a means for eliminating the background color and adverse effects of interfering substances.
It is an object of the present invention to overcome the aforesaid deficiencies in the cited art.
It is another object of the present invention to provide a method for estimating white blood cell count in a biological fluid.
It is a further object of the present invention to provide a device for estimating white blood cell count in a biological fluid.
It is another object of the present invention to provide a method and device for diagnosing mastitis.
It is still another method of the present invention to provide a method for estimating white blood cell count using a color reaction.
According to the present invention, white blood cell count in a biological fluid sample containing interfering substances can be obtained by capturing target cells using a membrane, and washing away interfering substances prior to the measurement step. This method and device can be used for assaying a wide variety of biological samples, such as blood, milk, urine, saliva, and sweat. Saliva can be monitored for white blood cell levels in order to detect dental problems, including infections in the gums and related parts of the mouth. Additionally, perspiration, or sweat, contains white blood cells, and thus may be monitored for levels of white blood cells present.
The method of the present invention comprises the following steps:
a. separating white blood cells from interfering substances from a biological fluid sample using a retainer which selectivity retains white blood cells;
b. removing interfering substances, such as red blood cells, enzymes, enzyme inhibitors, proteins, and lipids, from the retaining substrate, leaving only white blood cells, by washing with a washing solution containing a buffer and optional additives in water or an aqueous solution of a polar organic liquid.
c. reading, by eye or by instrument, the result of a color reaction in which an enzyme present in the white blood cells cleaves a chromogenic substrate which produces a water insoluble dye.
The chromogenic substrate can be either immobilized on the membrane or can be used as a component of the washing solution.
In a preferred embodiment, the enzyme is esterase and the chromogenic substrate is an ester.
The device of the present invention comprises:
a. a cover which has an opening for the application of a sample and a wash solution;
b. a white blood cell retainer that optionally has a dye substrate immobilized thereon;
c. an absorption layer that wicks and takes up all excess washing solution flowing past the sample;
d. a wash solution that contains a buffer, an optional reaction accelerator, and an optional dye substrate.